Microfluidic chip

Abstract

A microfluidic chip is disclosed. The microfluidic chip may include: at least one transparent substrate; at least one transparent cover; and at least one cavity defined between the at least one transparent substrate and the at least one transparent cover. In some embodiments, the at least one transparent cover and the at least one transparent substrate may include a material characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm-1 range.

Description

PHNT-P-002-PCTMICROFLUIDIC CHIPCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 730,987, filed December 12, 2024, the contents of which are all incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The present invention relates generally to methods of microfluidic chips. More specifically, the present invention relates to microfluidic chips for analyzing body fluids.BACKGROUND OF THE INVENTION

[0003] Microfluidic technology involves the manipulation of fluids at a microscale, typically within channels that are tens to hundreds of micrometers in dimension. Microfluidic chips, also known as lab-on-a-chip devices, integrate multiple laboratory functions on a single chip, enabling the automation and miniaturization of complex biochemical processes. These chips are fabricated using various materials such as glass, polymers, silicon and the like.

[0004] Microfluidic chips have revolutionized the fields of chemistry, biology, and medicine by providing a platform for high-throughput screening, diagnostics, and analytical applications. The ability to precisely control and manipulate small volumes of fluids allows for rapid and efficient reactions, reduced reagent consumption, and enhanced sensitivity and specificity in detection.

[0005] Traditional methods of blood analysis, such as flow cytometry and hematology analyzers, have been the cornerstone of clinical diagnostics for several decades. These methods involve the use of sophisticated instruments to analyze various components of blood, including red blood cells, white blood cells, and platelets. Flow cytometry, in particular, is a powerful technique that allows for the detailed analysis of cell populations based on their physical and chemical characteristics.

[0006] Raman spectroscopy is a powerful analytical technique that has found significant applications in the field of cell analysis. Raman spectroscopy offers several advantages. It is a label-free technique, meaning that it does not require the use of fluorescent or radioactivePHNT-P-002-PCT labels to detect specific molecules. This reduces the complexity and cost of sample preparation and eliminates potential artifacts introduced by labeling. Additionally, Raman spectroscopy is non-destructive, allowing for the analysis of live cells without compromising their viability.

[0007] Therefore, the integration of microfluidic chips as carriers for blood cells or other body fluids, in Raman spectroscopy offers several significant advantages.SUMMARY OF THE INVENTION

[0008] Some aspects of the invention may be related to a microfluidic chip comprising: at least one transparent substrate; at least one transparent cover; and at least one cavity defined between the at least one transparent substrate and the at least one transparent cover. In some embodiments, the at least one transparent cover and the at least one transparent substrate may include a material characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm'1range.

[0009] In some embodiments, the at least one transparent cavity may be characterized by a thickness of between 0.05 to 2 mm. In some embodiments, the least one cavity a depth of between 3 to 500 pm. In some embodiments, the depth variation is at most 5% from the total depth of the cavity.

[0010] In some embodiments, the at least one cavity may have a volume of between 0.1 to 100 pL. In some embodiments, the at least one cavity may have a volume of between 1 to 5 pL. In some embodiments, the material may be CaF2. In some embodiments, the material is quartz.

[0011] In some embodiments, the microfluidic chip may further include at least one adhesive layer located between the at least one transparent substrate and the at least one transparent cover, and wherein each cavity’s walls are defined by a conduit in the adhesive layer. In some embodiments, the at least one adhesive layer may have a thickness of between 3 to 500 pm.

[0012] In some embodiments, the microfluidic chip may further include: a substrate frame holding one transparent substrate; and a cover frame holding one transparent cover, wherein when attached to each other; (a) a gap is formed between the transparent substrate and the transparent frame, and (b) the cavity’s walls are defined by at least one of: the substrate frame and the cover frame.PHNT-P-002-PCT

[0013] In some embodiments, the at least one cavity is defined by an adjustable wall configured to adjust the volume of the at least one cavity. In some embodiments, the microfluidic chip may further include: for each cavity, an inlet configured to provide fluid to the cavity, and an outlet configured to extract the fluid from the cavity. In some embodiments, the microfluidic chip may further include: at least one inlet valve, in fluid connection with the inlet, for controlling the provision of the fluid to the cavity. In some embodiments, the microfluidic chip may further include: at least one outlet valve, in fluid connection to the outlet for controlling the extraction of the fluid from the cavity.

[0014] Some additional aspects of the invention may be a system comprising two or more microfluidic chips according to any one of the embodiments disclosed herein.

[0015] Some additional aspects of the invention may include the microfluidic chip according to any one of the embodiments disclosed herein; an inlet valve, in fluid connection with the inlet, for controlling the provision of the fluid to the cavity; an outlet valve, in fluid connection to the outlet for controlling the extraction of the fluid from the cavity; and a controller configured to control the inlet valve and the outlet valve.

[0016] In some embodiments, the controller may be configured to control the provision of a predetermined amount of a first type of fluid to the cavity. In some embodiments, the controller may be configured to control a continuous provision and extraction of at least a second type of fluid to and from the cavity. In some embodiments, the system may further comprise a multiplexer in fluid connection with at least the inlet valve, for interchanging between fluids. In some embodiments, the system may further comprise a mixer for mixing tow or more liquid.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0018] Figs. 1A and IB are illustrations of a perspective view and an exploded view of a microfluidic chip according to some embodiments of the invention;

[0019] Figs. 2 A and 2B are illustrations of a perspective view and an exploded view of a system comprising a microfluidic chip according to some embodiments of the invention;PHNT-P-002-PCT

[0020] Figs. 3A and 3B are illustrations of a perspective view and an exploded view of another microfluidic chip according to some embodiments of the invention;

[0021] Fig. 3C is an illustration of a system comprising the microfluidic chip of Figs. 3A and 3B according to some embodiments of the invention;

[0022] Figs. 4A and 4B are illustrations of a perspective view and an exploded view of another microfluidic chip according to some embodiments of the invention;

[0023] Figs. 5A and 5B are illustrations of a perspective view and an exploded view of another microfluidic chip according to some embodiments of the invention;

[0024] Fig. 6 is a block diagram, depicting a system comprising a microfluidic chip according to some embodiments of the invention; and

[0025] Fig. 7 is an image of a single layer of blood cells inside a microfluidic chip according to some embodiments of the invention.

[0026] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0027] One skilled in the art will realize the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. Scope of the invention is thus indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

[0028] The integration of microfluidic chips as carriers for bloody fluids (e.g., blood, urine, milk, saliva, sweat, and the like) in Raman spectroscopy may offer several significant advantages. For example, microfluidic chips may enable a precise control and manipulation of small volumes of blood, urine, or milk, allowing for the efficient handling and analysis of individual particle such as, cells or small cell populations, kidney stones, and the like. This precision may reduce sample consumption and eliminating the need of expensive reagents, making the process more cost-effective. In another example, the micro-scale environmentPHNT-P-002-PCT of the chip may provide a controlled and stable setting for Raman spectroscopy, enhancing the reproducibility and accuracy of the measurements. Furthermore, microfluidic chips according to embodiments of the invention may allow to use a fully automated measuring system, unlike the conventional flowcytometers which require manual work.

[0029] As used herein, a body fluid, may refer to body fluids that contain cells or mineral particles that can be inspected with Raman spectroscopy. For example, blood, saliva, urine, milk, sweat, and the like.

[0030] In some embodiments, the use of transparent materials such as calcium fluoride (CaF2) in the construction of the chips may ensure minimal interference with the Raman signal, thereby improving the sensitivity and specificity of the analysis. Additionally, the integration of microfluidic chips with automated fluid handling systems facilitates high- throughput screening and real-time monitoring of cellular / particle responses, significantly speeding up the analytical process. This combination of microfluidic technology and Raman spectroscopy may offer a powerful platform for detailed, rapid, and cost-effective body fluid analysis, with potential applications in clinical diagnostics, personalized medicine, and biomedical research.

[0031] Reference is now made to Figs. 1A and IB which are illustrations of a perspective view and an exploded view of a microfluidic chip according to some embodiments of the invention. A microfluidic chip 10 may include at least one transparent substrate 12 and at least one transparent cover 14. The microfluidic chip 10 may include at least one cavity 15 defined between the transparent substrate 12 and the transparent cover 14. The transparent cover 14 and the transparent substrate 12 may be made of a material characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm-1 range. In a nonlimiting example, the material may be calcium fluoride (CaF2).

[0032] In some embodiments, transparent substrate 12 may be designed to provide the necessary mechanical support for the microfluidic chip 10. Transparent substrate 12 may have sufficient thickness to maintain structural integrity while being thin enough to allow for efficient light transmission. The thickness of the transparent substrate 12 can vary depending on the specific design requirements, but typically ranges from 0.5 to 10 mm. In the nonlimiting example illustrated in Figs. 1A and IB transparent substrate 12 is a rectangular plate, however, any other shape is within the scope of the invention.PHNT-P-002-PCT

[0033] In some embodiments, transparent cover 14 may be characterized by a thickness of between 0.05 to 2 mm, for example, 0.1 mm, 0.2 mm, 0.4 mm, 0.5 mm, 0.8 mm, 1 mm, 1.2 mm, 1.5 mm, 1.8 mm, 2 mm, and any value or range in between. In the nonlimiting example of Fig. IB, transparent cover 14 is shaped as a flat disc, however, any suitable shape characterized by a thickness of between 0.05 to 2 mm is within the scope of the invention.

[0034] In some embodiments, cavity 15 may have a depth of between 3 to 500 pm, for example, 3 pm, 5 pm, 10 pm, 20 pm, 50 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, and any value or range in between. In some embodiments, microfluidic chip 10 further comprises an adhesive layer 16 located between the transparent substrate 12 and the transparent cover 14, and cavity 15 walls may be defined by a conduit, in the adhesive layer 16. Various geometries of such conduit are shown in Figs. IB and 2B. Therefore, in some embodiments, the thickness of adhesive layer 16 may be similar to the depth of cavity 15, of between 3 to 500 pm.

[0035] In some embodiments, adhesive layer 16 may include or may be made of microfluidic diagnostic tape, for example, 3M™ 9969 Adhesive Transfer Tape. Such an adhesive layer may be characterized to be non-hemolytic, non-toxic to mammalian cell, low extractable, transparent, low autofluorescence, and the like. In some embodiments, cavity 16 may be formed in adhesive layer 16 using any known method, some nonlimiting examples may include, micro-machining, laser cutting, lase ablation, and the like.

[0036] In some embodiment, cavity 15 may have a volume of between 0.1 to 100 pL, and more specifically, between 1 to 5 pL. For example, cavity 15 may have a volume of 0.1 pL, 0.5 pL, 1 pL, 1.5 pL, 2 pL, 3 pL, 4 pL, 5 pL, 7 pL, 8 pL, 10 pL, 20 pL, 30 pL, 40 pL, 50 pL, 60 pL, 70 pL, 80 pL, 90 pL, 100 pL and any value or range in between.

[0037] In some embodiments, microfluidic chip 10 may also include an inlet 11 configured to provide fluid to cavity 15 and an outlet 13 configured to extract the fluid from cavity 15. Inlet 11 and outlet 13 are positioned to facilitate the flow of fluids through cavity 15, for example, to enable efficient handling and analysis of body fluid within the microfluidic chip 10. In the nonlimiting example illustrated in Figs. 1 A and IB, inlet 11 and outlet 13 are holes in transparent substrate 12, overlapping, defined within the area of cavity 15.

[0038] The use of transparent materials, for fabricating transparent substrate 12 and transparent cover 14, for example, CaF2 or quartz may ensure minimal interference with the Raman signal, thereby improving the sensitivity and specificity of the analysis. In somePHNT-P-002-PCT embodiments, this material may also be transparent in the visible light spectrum, as well as, being characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm-1range.

[0039] In some embodiments, transparent substrate 12 and transparent cover 14 may be fabricated using any suitable method, for example, machining, laser cutting, casting, and the like.

[0040] In some embodiments, microfluidic chip 10 may include more than one cavity 15 covered by more than one transparent cover 14. In the nonlimiting example illustrated in Figs. 1A and IB, microfluidic chip 10 includes one transparent substrate, 3 cavities 15 covered by 3 transparent covers 14. Microfluidic chip 10, of Figs. 1 A and IB may also have 3 inlets 11, and 3 outlets 13. As should be understood by the one skilled in the art, microfluidic chip 10 may include any number of substrates, cavities, and transparent covers, for example, 1, 2, 3, 4, 5, 6, 10, 50 or more. Such a design may allow performing parallel analysis of different samples, performing a cleaning procedure to some cavities while imaging and analyzing (e.g., by Raman Spectroscopy) other cavities of the same microfluidic chip 10.

[0041] In some embodiments, the material selected fortransparent substrate 12, transparent over 14, and / or adhesive layer 16 may be chemically inert and resistant to the reagents and solvents used in the microfluidic processes. For example, cleaning microfluidic chip 10 may be conducted by flushing cavity 15 with 3 different solutions, first with cleaning solutions such as the commercially available CONTRAD 70, followed by flushing cavity 15 with sodium hypochlorite solution at a constitution of between 11 to 15 %, and finally flushing with flow clean In Vitro Diagnostic (IVD) solution.

[0042] Reference is now made to Figs. 2A and 2B, which are illustrations of a perspective view and an exploded view of a system comprising a microfluidic chip according to some embodiments of the invention. A system 100 includes a microfluidic chip 10, an inlet valve 30, an outlet valve 40, and a holder 90. In some embodiments, microfluidic chip 10 may be substantially the same as microfluidic chip 10 discussed with respect to Figs. 1A and IB. In some embodiments, microfluidic chip 10 may vary only in the geometry of cavity 15. In some embodiments, all cavities 15 of microfluidic chip 10 may be identical. In some embodiments, microfluidic chip 10 may include two or more cavities 15 having at least two different geometries.PHNT-P-002-PCT

[0043] In some embodiments, system 100 may further include inlet valve 30 in fluid connection with inlet 11, for controlling the provision of the fluid to cavity 15, and outlet valve 40 in fluid connection with outlet 13, for controlling the extraction of the fluid from cavity 15. In some embodiments, inlet valve 30 and outlet valve 40 may be integrated with microfluidic chip 10 in order to enhance its functionality and control fluid flow precisely. Potential valves that may be used together with the microfluidic chip 10 include solenoid valves, pneumatic valves, piezoelectric valves, and the like.

[0044] Solenoid valves are electrically controlled and can provide rapid and precise control of fluid flow, making them suitable for automated systems. Pneumatic valves, which are operated by compressed air, offer robust performance and can handle a wide range of fluid types and pressures. Piezoelectric valves, on the other hand, utilize piezoelectric materials to control fluid flow with high precision and low power consumption, making them ideal for applications requiring fine control and minimal energy usage. The selection of the appropriate valve type may depend on the specific requirements of the application, such as the desired flow rate, pressure, and level of automation. Integrating these valves with the microfluidic chip 10 may allow for efficient and accurate manipulation of fluids, enabling high-throughput screening, real-time monitoring, and precise control of the conditions.

[0045] In some embodiments, holder 90 may be any platform configured to hold microfluidic chip 10. Holder 90, may be designed to secure microfluidic chip 10, therefore, may have to meet several requirements to ensure seamless integration with a Raman spectroscopy system. In some embodiments, holder 90 may provide precise alignment and stable positioning of the microfluidic chip 10 to ensure accurate and reproducible Raman measurements. This stability may be crucial to prevent movement or vibrations that could affect the quality of the Raman spectra. In some embodiments, holder 90 may be constructed from materials that do not interfere with the Raman signal, such as non-fluorescent and nonRaman active materials, to avoid introducing background noise or artifacts into the measurements.

[0046] In some embodiments, holder 90 may allow for easy access to microfluidic chip 10 for fluidic connections, including the inlet and outlet valves, while maintaining a secure seal to prevent leaks. It should also be compatible with the optical setup of the Raman spectroscopy system, ensuring that the laser beam can be accurately focused on the desired regions of microfluidic chip 10. Furthermore, holder 90 may facilitate efficient heatPHNT-P-002-PCT dissipation to prevent any thermal effects that could impact the Raman analysis. By meeting these requirements, holder 90 may ensure that the microfluidic chip 10 can be effectively integrated with a Raman spectroscopy system, enabling high-quality, reliable, and precise cellular / particle analysis.

[0047] Reference is now made to Figs. 3 A and 3B, which are illustrations of a perspective view and an exploded view of another microfluidic chip according to some embodiments of the invention. A microfluidic chip 20 may include a transparent substrate 22, a transparent cover 24 and at least one flat cavity 29 defined between transparent substrate 22 and transparent cover 24.

[0048] In some embodiments, transparent substrate 22 may include a plate or a disc made from a material characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm-1 range. In some embodiments, the material may also be transparent in the visible wavelength range. In the nonlimiting example illustrated in Figs. 3A and 3B, transparent substrate 22 is a round disc, however, other shapes are within the scope of the invention (e.g., rectangular, diagonal, hexagonal, trapezoid, etc.). In some embodiments, the thickness of substrate 22 may be between 0.5 to 10 mm, and any value or range in between.

[0049] In some embodiments, transparent substrate 22 may be held by a substrate frame 23. Substrate frame 23 may include a recess or groove that matches the dimensions and shape of the transparent substrate 22, allowing it to fit snugly and securely. This precise fit prevents any lateral or vertical movement of the substrate 22, which is crucial for maintaining the integrity of the microfluidic channels and ensuring accurate and reproducible measurements during Raman spectroscopy.

[0050] In some embodiments, transparent cover 24 may include a plate or a disc made from a material characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm-1 range. In some embodiments, the material may also be transparent in the visible wavelength range.

[0051] In some embodiments, transparent cover 24 may be characterized by a thickness of between 0.05 to 2 mm, for example, 0.1 mm, 0.2 mm, 0.4 mm, 0.5 mm, 0.8 mm, 1 mm, 1.2 mm, 1.5 mm, 1.8 mm, 2 mm and any value or range in between. In the nonlimiting example of Fig. IB, transparent cover 24 is shaped as a flat disc, however, any suitable shape characterized by a thickness of between 0.05 to 2 mm is within the scope of the invention.PHNT-P-002-PCT

[0052] In some embodiments, the design of the substrate frame 23 may also facilitate easy assembly and disassembly of the microfluidic chip 20, allowing for convenient cleaning, maintenance, and replacement of transparent substrate 22 if necessary. This modularity may be particularly important in applications requiring frequent changes of the microfluidic environment or when different substrates need to be tested.

[0053] In some embodiments, microfluidic chip 20 may further include a cover frame 25 holding transparent cover 24. Cover frame 25 may be designed to securely hold the transparent cover 24 in place, ensuring its stability and proper alignment within the microfluidic chip 20. Cover frame 25 may include a recess or groove that matches the dimensions and shape of the transparent cover 24, allowing it to fit snugly and securely. This precise fit prevents any lateral or vertical movement of the cover 24, which is crucial for maintaining the integrity of the microfluidic channels and ensuring accurate and reproducible measurements during Raman spectroscopy.

[0054] In some embodiments, substrate frame 23 and cover frame 24 may both be made from a metallic alloy, or from any other suitable material.

[0055] In some embodiments, when substrate frame holding substrate 22 is attached to cover frame 25 holding cover 24, a gap is formed between transparent substrate 22 and transparent cover 24, thereby forming cavity 29. In some embodiments, cavity 29 walls may be defined by at least one of: substrate frame 23 and cover frame 25. Optionally, substrate frame 23 may be attached to cover frame 25 via a seal ring 26. Substrate frame 23 and cover frame 25 may be screwed to each other or to seal ring 26, glued to each other or to seal ring 26, clicked to each other or to seal ring 26, and the like.

[0056] In some embodiments, cavity 29 may have a depth of between 3 to 500 pm, for example, 3 pm, 5 pm, 10 pm, 20 pm, 50 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm and any value or range in between. In some embodiment, cavity 29 may have a volume of between 0.1 to 10 pL, and more specifically, between 1 to 5 pL. For example, cavity 15 may have a volume of 0.1 pL, 0.5 pL, 1 pL, 1.5 pL, 2 pL, 3 pL, 4 pL, 5 pL, 7 pL, 8 pL, 10 pL, and any value or range in between.

[0057] In some embodiments, in order to ensure a precise alignment of the depth of cavity 29, a spacer may be placed between transparent substrate 22 and transparent cover 24 during the assembly. The spacer may have a thickness of between 3 to 500 pm and may be removedPHNT-P-002-PCT after the fixing of transparent substrate 22 and transparent cover 24 to their respective frames.

[0058] In some indictments, cavity 29 of chip 20 may include an adjustable wall configured to adjust the volume of cavity 29. Therefore, chip 20 may include any suitable mechanisms (not illustrated) for adjusting the volume of cavity 29. Some nonlimiting examples for such mechanisms may include, a screw, micrometer screws, precision guides, or automated actuators and the like.

[0059] Reference is now made to Fig. 3C, which is an illustration of a system comprising the microfluidic chip of Figs. 3 A and 3B according to some embodiments of the invention. System 100 may include microfluidic chip 20 (or microfluidic chip 10), holder 90 and an alignment mechanism 95. Alignment mechanism 95 may be an optional but highly beneficial component that can be integrated into system 100 to enhance the precision and reliability of microfluidic chip 20 or 10 during analytical procedures. The primary function of alignment mechanism 95 may be to ensure that the microfluidic chip 20 or 10 is accurately positioned relative to the optical components of the system.

[0060] In some embodiments, alignment mechanism 95 may include features such as micrometer screws, precision guides, or automated actuators that allow for fine adjustments in the positioning of microfluidic chip 20 or 10. These adjustments may be made in multiple dimensions, including lateral (X and Y axes) and vertical (Z axis) directions, ensuring that the microfluidic chip 20 is perfectly aligned with the laser beam and detection optics of the Raman spectroscopy system. This precise alignment is crucial for obtaining high-quality, reproducible Raman spectra and for ensuring accurate fluid flow through the microfluidic channels.

[0061] Reference is now made to Figs. 4A and 4B and further to Figs 5 A and 5B which are illustrations of a perspective view and an exploded view of additional microfluidic chips according to some embodiments of the invention. A microfluidic chip 10a and / or 10b may include at least one transparent substrate 12a and / or 12b and at least one transparent cover 14a and / or 14b. Microfluidic chip 10a may include two linear cavities 15a defined between the transparent substrate 12a and the transparent cover 14a. The two linear cavities 15a may be in fluid connection with inlet I la and outlet 13a. The sample may be inserted from inlet 1 la to be exit (by washing) from exit 13a.PHNT-P-002-PCT

[0062] Microfluidic chip 1 Ob, illustrated in Figs. 5 A and 5B, may include two pairs of linear cavities 15b defined between the transparent substrate 12b and the transparent cover 14b. The two pairs of linear cavities 15b may each be in fluid connection with one inlet 1 lb and one outlet 13b. Samples may be inserted from inlets 1 lb to be exit (by washing) from exits 13b.

[0063] Inlets I la and 11b and outlets 13a and 13b may located at the side faces of transparent covers 14a, 14b and the transparent substrate 12a, 12b.

[0064] The transparent covers 14a, 14b and the transparent substrate 12a, 12b may be made of a material characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm-1 range. In a nonlimiting example, the material may be calcium fluoride (CaF2).

[0065] In some embodiments, transparent substrates 12a, 12b may be designed to provide the necessary mechanical support for the microfluidic chips 10a and 10b. Transparent substrate 12a and 12b may have sufficient thickness to maintain structural integrity while being thin enough to allow for efficient light transmission. The thickness of the transparent substrate 12a and / or 12b can vary depending on the specific design requirements, but typically ranges from 0.5 to 10 mm.

[0066] In some embodiments, transparent covers 14a and / or 14b may be characterized by a thickness of between 0.05 to 2 mm, for example, 0.1 mm, 0.2 mm, 0.4 mm, 0.5 mm, 0.8 mm, 1 mm, 1.2 mm, 1.5 mm, 1.8 mm, 2 mm, and any value or range in between.

[0067] In some embodiments, any one of cavities 15a and 15b may have a depth of between 3 to 500 pm, for example, 3 pm, 5 pm, 10 pm, 20 pm, 50 pm, 100 pm, 150 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 600 pm, 1000 pm, 1500 pm and any value or range in between.

[0068] In some embodiments, cavities 15a and 15b may have a volume of between 0.1 to 100 pL, and more specifically, between 1 to 5 pL. For example, cavity 15 may have a volume of 0.1 pL, 0.5 pL, 1 pL, 1.5 pL, 2 pL, 3 pL, 4 pL, 5 pL, 7 pL, 8 pL, 10 pL, 20 pL, 30 pL, 40 pL, 50 pL, 60 pL, 70 pL, 80 pL, 90 pL, 100 pL and any value or range in between.

[0069] In some embodiments, linear cavities 15a and 15b may provide homogenous flow of the samples.

[0070] The use of transparent materials, for fabricating transparent substrate 12a and / or 12b and transparent cover 14a and / or 14b, for example, CaF2 or quartz may ensure minimal interference with the Raman signal, thereby improving the sensitivity and specificity of thePHNT-P-002-PCT analysis. In some embodiments, this material may also be transparent in the visible light spectrum, as well as, being characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm-1range.

[0071] In some embodiments, microfluidic chips 10a and 10b may further include a reference element 18 attached to / embedded in any of transparent substrates 12a, 12b and transparent covers 14a and 14b, for example, in cavity 19. In a nonlimiting example, the reference element may be a Si chip designed to allow the calibration of the Raman Spectrometer, with known spectrum (e.g., the Si spectrum). This calibration may allow assessing the effect of environmental conditions such as temperature, humidity, and the like on the Raman spectroscopy.

[0072] In some embodiments, element 18 may be or may include additional sensors, such as temperature sensor, or any other electronic component, as required for specific designs.

[0073] In some embodiments, transparent substrate 12a and / or 12b and transparent cover 14a and / or 14b may be fabricated using any suitable method, for example, machining, laser cutting, casting, and the like.

[0074] In some embodiments, the volume of cavities, 15, 25, 15a, and 15b, may be known and calibrated, as to allow conducting an absolute count of cell / particles, for example, to result in the amount of cells / particles per microliter, as discloses and discuss in the Example section below. In some embodiments, the depth (or parallelism) of cavities 15, 25, 15a, and 15b may vary in at most 10%, for example, at most 8%, and most 7% at most 5%, and most 4% and most 3% at most 1 % and any value or range in between. In some nonlimiting example, the depth of cavities, 15, 25, 15a, and 15b, may be between 40 to 50 pm, with depth variation of ±l m. In another nonlimiting example, for kidney dusting residues, the depth of cavities, 15, 25, 15a, and 15b, may be between 500 to 600 pm, with depth variation of ±10pm.

[0075] In some embodiments, transparent substrate 12, 22, 12a and / or 12b and transparent cover 14, 24, 14a and / or 14b may be polished to an optical grade accoridng to standard ISO 10110, of at least at precision quality or at high / laser quality.

[0076] Reference is now made to Fig. 6, which illustrates a block diagram of a system 100 comprising multiple microfluidic chips 10 / 20 according to some embodiments of the invention. System 100 is designed to facilitate the precise control and manipulation of fluids within the microfluidic chips for various analytical applications.PHNT-P-002-PCT

[0077] System 100 may include one or more microfluidic chips 10 / 20, each may be equipped with an inlet valve 30 and / or an outlet valve 40. The inlet valves 30 are configured to control the provision of fluids to the cavities within the microfluidic chips 10 / 20, while outlet valves 40 alone or in combination with inlet valve 30 are controlled to reduce stabilization times of the cells inside the cavity. This configuration may allow for efficient handling and analysis of samples within the microfluidic chips. During the insertion of the sample or any other liquid (e.g., cleaning solutions) inlet valves 30 and outlet valves 40 are open. In some embodiments, in order to rapidly stabilize the sample inside cavities 16 or 29, at least one of valves 30 or 40 is closed. In some embodiments, at least one of valves 30 or 40 may be modulated (e.g., alternating opening and closure of the valve).

[0078] System 100 may further include a controller 50, which is configured to control the operation of the inlet valves 30 and outlet valves 40. Controller 50 may be programmed to manage the flow of fluids through the microfluidic chips 10 / 20, ensuring precise and accurate fluid handling. Controller 50 may also be connected to a pump 60, which may provide the necessary pressure to drive the fluids through the system.

[0079] In some embodiments, pump may be any precise pump that may provide an accurate control of very low amounts of fluids, for example, from 0.5 pL to several ml. Some nonlimiting examples may include, syringe pumps that uses a motor-driven plunger to precisely control the volume of fluid dispensed from a syringe. They offer high accuracy and are commonly used in applications requiring precise dosing and flow control. Additional nonlimiting examples may include piezoelectric pumps which use piezoelectric materials that deform when an electric field is applied, creating a pumping action. They are compact and can provide precise control of fluid flow.

[0080] In some embodiments, the system 100 includes a first fluid source 70 and a second fluid source 80. These fluid sources can supply different types of fluids to the microfluidic chips 10 / 20, enabling various analytical procedures. For example, first fluid source 70 may include the body fluid sample, and second fluid source 80 may include cleaning solutions. As should be understood by the one skilled in the art, system 100 may include more than one sample source and more than one (e.g., 3) cleaning solutions source. The controller 50 can be configured to control the provision of a predetermined amount of a first type of fluid from the first fluid source 70 to the cavities within the microfluidic chips 10 / 20.PHNT-P-002-PCTAdditionally, the controller 50 can manage the continuous provision and extraction of at least a second type of fluid from the second fluid source 80 to and from the cavities.

[0081] System 100 may also include a multiplexer 75 in fluid connection with at least the inlet valves 30 and at least first fluid source 70 and second fluid source 80, allowing for the interchange between different fluids. This feature enhances the flexibility and versatility of the system, enabling it to perform a wide range of analytical procedures. System 100 may further include a mixer 76 for mixing two or more fluids, prior to the introduction of the fluids to cavities 16 or 29.

[0082] In a nonlimiting example, the first fluid may be body fluid or a solution comprising body fluid (e.g., blood) material for inspection. In such a case, controller 50 may control pump 60 and inlet valve 30 to provide a precise amount of the first fluid for inspection (e.g., by Raman Spectroscopy) from first fluid source 70, while maintaining outlet valve 40 closed. At the end of the inspection, controller 50 may control the opening of both inlet valve 30 and outlet valve 40, and pump 60 to continuously provide one or more cleaning solutions for cleaning cavities 16 or 29. In some embodiments, multiplexer 75 may be controlled to switch between various liquid sources.

[0083] In some embodiments, controller 50 may control the provision of the body fluid or a solution comprising the body fluid material to at least some of microfluidic chips 10 / 20 of system 100 while controlling the provision of cleaning solutions to other microfluidic chips 10 / 20 of the same system 100.Examples

[0084] Reference is now made to Fig. 7 which is a microscopy image of a sample of blood cells placed inside microfluidic chip 10a having a depth of 40pm. The parallelism between transparent substrate 12a and transparent cover 14a, was at most I . These dimensions were chosen to ensure the formation of a single cells layer, such that the cells do not overlap with each other, thereby allowing cell count.

[0085] Unless explicitly stated, the method embodiments described herein are not constrained to a particular order or sequence. Furthermore, all formulas described herein are intended as examples only and other or different formulas may be used. Additionally, some of the described method embodiments or elements thereof may occur or be performed at the same point in time.PHNT-P-002-PCT

[0086] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents may occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

[0087] Various embodiments have been presented. Each of these embodiments may of course include features from other embodiments presented, and embodiments not specifically described may include various features described herein.

Claims

PHNT-P-002-PCTCLAIMS1. A microfluidic chip comprising: at least one transparent substrate; at least one transparent cover; and at least one cavity defined between the at least one transparent substrate and the at least one transparent cover, wherein the at least one transparent cover and the at least one transparent substrate comprise a material characterized by Raman Spectroscopy maxima outside of the 400 to 2000 cm'1range.

2. The microfluidic chip of claim 1, wherein the at least one transparent cavity is characterized by a thickness of between 0.05 to 2 mm.

3. The microfluidic chip of claim 1 or claim 2, wherein the least one cavity a depth of between 3 to 500 pm.

4. The microfluidic chip of claim 3, wherein the depth variation is at most 5% from the total depth of the cavity.

5. The microfluidic chip of any one of claims 1 to 4, wherein the at least one cavity has a volume of between 0.1 to 100 pL.

6. The microfluidic chip of claim 5, wherein the at least one cavity has a volume of between 1 to 5 pL.

7. The microfluidic chip of any one of claims 1 to 6, wherein the material is CaF2.

8. The microfluidic chip of any one of claims 1 to 7, wherein the material is quartz.

9. The microfluidic chip of any one of claims 1 to 8, further comprising at least one adhesive layer located between the at least one transparent substrate and the at least one transparent cover, and wherein each cavity’s walls are defined by a conduit in the adhesive layer.

10. The microfluidic chip of claim 9, wherein the at least one adhesive layer has a thickness of between 3 to 500 pm.

11. The microfluidic chip of any one of claims 1 to 8, further comprising: a substrate frame holding one transparent substrate; and a cover frame holding one transparent cover, wherein when attached to each other;PHNT-P-002-PCT a. a gap is formed between the transparent substrate and the transparent frame, and b. the cavity’s walls are defined by at least one of: the substrate frame and the cover frame.

12. The microfluidic chip of any one of claims 1 to 8, wherein the at least one cavity is defined by an adjustable wall configured to adjust the volume of the at least one cavity.

13. The microfluidic chip of any one of claims 1 to 10, further comprising: for each cavity, an inlet configured to provide fluid to the cavity, and an outlet configured to extract the fluid from the cavity.

14. The microfluidic chip of claim 12, further comprising at least one inlet valve, in fluid connection with the inlet, for controlling the provision of the fluid to the cavity.

15. The microfluidic chip of claim 13 or claim 14, further comprising at least one outlet valve, in fluid connection to the outlet for controlling the extraction of the fluid from the cavity.

16. A system comprising two or more microfluidic chips according to any one of claims 1 to 15.

17. A system comprising: the microfluidic chip according to claim 13; an inlet valve, in fluid connection with the inlet, for controlling the provision of the fluid to the cavity; an outlet valve, in fluid connection to the outlet for controlling the extraction of the fluid from the cavity; and a controller configured to control the inlet valve and the outlet valve.

18. The system of claim 17, wherein the controller is configured to control the provision of a predetermined amount of a first type of fluid to the cavity.

19. The system of claim 17, wherein the controller is configured to control a continuous provision and extraction of at least a second type of fluid to and from the cavity.PHNT-P-002-PCT20. The system according to any one of the claims 16 to 18, further comprising a multiplexer in fluid connection with at least the inlet valve, for interchanging between fluids.

21. The system according to any one of the claims 16 to 19, further comprising a mixer for mixing two or more liquids.

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